Inhibitory potency of R-region specific antisense oligonucleotides against in vitro DNA polymerization and template-switching reactions catalysed by HIV-1 reverse transcriptase.
Antisense oligonucleotides (AONs) targeted to the R-region near the 5'-LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong stop DNA and the first template-switch reaction catalysed by HIV-1 reverse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3'-terminal nucleotide, with either 2'-deoxy-D-ribose (DNA), 2'-deoxy-L-ribose (L), or arabinose (ARA) in this position. All three AONs hybridized to complementary 18 nt RNA (T(m) approximately 70 degrees C) and specifically interacted with the target RNA HIV-1 sequence at 37 degrees C. L was unable to serve as primer for RT-catalysed DNA polymerization, whereas priming from ARA was about 30% that noted with DNA. Each of the three AONs resulted in similar 85-95% decreases in the amount of full length (-) strong stop DNA and up to 75% decreases in the first template-switch reaction products formed by RT, implying that elongation of the AONs did not enhance the inhibitory activity in vitro. A concomitant increase in a truncated DNA product corresponding to polymerization termination at the 5'-end of the AON was noted, indicating that RT was unable to displace the AON. Interestingly, near maximal inhibition in vitro an AON:target RNA template ratio of 1:1 was noted. Our results confirm the validity of our in vitro system for the analysis of potential antisense oligonucleotide inhibitors, and suggest that antisense oligonucleotides directed to the R-region of HIV-1 RNA may be effective inhibitors of the initial stages of HIV-1 proviral DNA synthesis.
Borkow
G
G
Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
Arion
D
D
Noronha
A
A
Scartozzi
M
M
Damha
M J
MJ
Parniak
M A
MA
D001704:Biopolymers; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis*; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012091:Repetitive Sequences, Nucleic Acid; D013698:Templates, Genetic
D001704:Biopolymers; D016376:Oligonucleotides, Antisense; D004247:DNA; D054303:HIV Reverse Transcriptase